Atomic resolution structures of native copper nitrite reductase from Alcaligenes xylosoxidans and the active site mutant Asp92Glu

We provide the first atomic resolution (<1.20 A) structure of a copper protein, nitrite reductase, and of a mutant of the catalytically important Asp92 residue (D92E). The atomic resolution where carbon-carbon bonds of the peptide become clearly resolved, remains a key goal of structural analysis. Despite much effort and technological progress, still very few structures are known at such resolution. For example, in the Protein Data Bank (PDB) there are some 200 structures of copper proteins but the highest resolution structure is that of amicyanin, a small (12 kDa) protein, which has been resolved to 1.30 A. Here, we present the structures of wild-type copper nitrite reductase (wtNiR) from Alcaligenes xylosoxidans (36.5 kDa monomer), the "half-apo" recombinant native protein and the D92E mutant at 1.04, 1.15 and 1.12A resolutions, respectively. These structures provide the basis from which to build a detailed mechanism of this important enzyme.     

High resolution structural studies of mutants provide insights into catalysis and electron transfer processes in copper nitrite reductase

We present high-resolution crystal structures and functional analysis of T1Cu centre mutants of nitrite reductase that perturb the redox potential and the Cys130-His129 "hard-wired" bridge through which electron transfer to the catalytic T2Cu centre occurs. These data provide insight into how activity can be altered through mutational manipulation of the electron delivery centre (T1Cu). The alteration of Cys to Ala results in loss of T1Cu and enzyme inactivation with azurin as electron donor despite the mutant enzyme retaining full nitrite-binding capacity. These data establish unequivocally that no direct transfer of electrons occurs from azurin to the catalytic type 2 Cu centre. The mutation of the axial ligand Met144 to Leu increases both the redox potential and catalytic activity, establishing that the rate-determining step of catalysis is the intermolecular electron transfer from azurin to nitrite reductase.     

Crystallography with online optical and X-ray absorption spectroscopies demonstrates an ordered mechanism in copper nitrite reductase

Nitrite reductases are key enzymes that perform the first committed step in the denitrification process and reduce nitrite to nitric oxide. In copper nitrite reductases, an electron is delivered from the type 1 copper (T1Cu) centre to the type 2 copper (T2Cu) centre where catalysis occurs. Despite significant structural and mechanistic studies, it remains controversial whether the substrates, nitrite, electron and proton are utilised in an ordered or random manner. We have used crystallography, together with online X-ray absorption spectroscopy and optical spectroscopy, to show that X-rays rapidly and selectively photoreduce the T1Cu centre, but that the T2Cu centre does not photoreduce directly over a typical crystallographic data collection time. Furthermore, internal electron transfer between the T1Cu and T2Cu centres does not occur, and the T2Cu centre remains oxidised. These data unambiguously demonstrate an ‘ordered’ mechanism in which electron transfer is gated by binding of nitrite to the T2Cu. Furthermore, the use of online multiple spectroscopic techniques shows their value in assessing radiation-induced redox changes at different metal sites and demonstrates the importance of ensuring the correct status of redox centres in a crystal structure determination. Here, optical spectroscopy has shown a very high sensitivity for detecting the change in T1Cu redox state, while X-ray absorption spectroscopy has reported on the redox status of the T2Cu site, as this centre has no detectable optical absorption.     

Cytoplasmic expression of the Achromobacter xylosoxidans blue copper nitrite reductase in Escherichia coli and characterisation of the recombinant protein

The gene of the Achromobacter xylosoxidans (DSM 2402) blue copper-containing nitrite reductase was amplified using the polymerase chain reaction. DNA sequence analysis reveals that the amino acid sequence is identical to those of the GIFU1051 and the NCIMB11015 A. xylosoxidans nitrite reductases. The gene encoding the mature coding region for DSM 2402 nitrite reductase was cloned into a pET-vector, overexpressed in the cytoplasm of Escherichia coli BL21(DE3), and the expressed holoprotein was purified to apparent homogeneity by cation-exchange chromatography. The recombinant blue copper-containing nitrite reductase was obtained in high yields of 70mgL(-1) of culture. The specific catalytic activity as well as the electronic absorption and electron paramagnetic resonance spectra agree with corresponding data for the native protein. Mass spectroscopic analysis of the recombinant nitrite reductase gave a molecular weight of 36659.1Da for the apo-protein monomer, in agreement with the expected molecular mass based on the amino acid sequence.     

Cyanide binding to cd(1) nitrite reductase from Pseudomonas aeruginosa: role of the active-site His369 in ligand stabilization

Cyanide binding to fully reduced Pseudomonas aeruginosa cd(1) nitrite reductase (Pa cd(1) NiR) has been investigated for the wild-type enzyme and a site-directed mutant in which the active-site His369 was replaced by Ala. This mutation reduces the affinity toward cyanide (by approximately 13-fold) and especially decreases the rate of binding of cyanide to the reduced d(1) heme (by approximately 100-fold). The crystal structure of wild-type reduced Pa cd(1) NiR saturated with cyanide was determined to a resolution of 2.7 A. Cyanide binds to the iron of the d(1) heme, with an Fe-C-N angle of 168 degrees for both subunits of the dimer and only His369 is within hydrogen bonding distance of the nitrogen atom of the ligand. These results suggest that in Pa cd(1) NiR the invariant distal residue His369 plays a dominant role in controlling the binding of anionic ligands and allow the discussion of the mechanism of cyanide binding to the wild-type enzyme.     

Structural and functional characterization of the Geobacillus copper nitrite reductase: Involvement of the unique N-terminal region in the interprotein electron transfer with its redox partner

The crystal structures of copper-containing nitrite reductase (CuNiR) from the thermophilic Gram-positive bacterium Geobacillus kaustophilus HTA426 and the amino (N)-terminal 68 residue-deleted mutant were determined at resolutions of 1.3 Å and 1.8 Å, respectively. Both structures show a striking resemblance with the overall structure of the well-known CuNiRs composed of two Greek key β-barrel domains; however, a remarkable structural difference was found in the N-terminal region. The unique region has one β-strand and one α-helix extended to the northern surface of the type-1 copper site. The superposition of the Geobacillus CuNiR model on the electron-transfer complex structure of CuNiR with the redox partner cytochrome c₅₅₁ in other denitrifier system led us to infer that this region contributes to the transient binding with the partner protein during the interprotein electron transfer reaction in the Geobacillus system. Furthermore, electron-transfer kinetics experiments using N-terminal residue-deleted mutant and the redox partner, Geobacillus cytochrome c₅₅₁, were carried out. These structural and kinetics studies demonstrate that the region is directly involved in the specific partner recognition.